Hepatitis B virus polymerase suppresses translation of pregenomic RNA via a mechanism involving its interaction with 5′ stem–loop structure

ABSTRACT We recently identified three nuclear proteins (p45, p39, and p26) that bind to a 91-nucleotide (nt) RNA element between nt 1243 and 1333 in hepatitis B virus (HBV) RNA, and we showed that these proteins and HBV RNA are regulated coordinately by gamma interferon and tumor necrosis factor alpha. Purification and sequence analysis of tryptic peptides obtained from p39 revealed sequence homology to the mouse La protein. Immunoprecipitation experiments showed that p45, p39, and p26 were recognized by anti-La-specific antiserum, indicating that p45 is the full-length La protein and that p39 and p26 are likely to be proteolytic La cleavage products. Furthermore, in competition experiments we found that all three La proteins bind, in a phosphorylation-dependent manner, to the same predicted stem-loop structure located between nt 1275 and 1291 of HBV, withKd s of approximately 1.0 nM. Collectively, these results support the notion that the La protein may contribute to HBV RNA stability, constitutively and in response to inflammatory cytokines.

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N6-methyladenosine modification of hepatitis B virus RNA differentially regulates the viral life cycle

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1808319115 ◽

2018 ◽

Vol 115 (35) ◽

pp. 8829-8834 ◽

Cited By ~ 44

Author(s):

Hasan Imam ◽

Mohsin Khan ◽

Nandan S. Gokhale ◽

Alexa B. R. McIntyre ◽

Geon-Woo Kim ◽

...

Keyword(s):

Life Cycle ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Reverse Transcription ◽

Mutational Analysis ◽

Regulatory Function ◽

Messenger Rnas ◽

Stem Loop ◽

B Virus ◽

A Site

N6-methyladenosine (m6A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m6A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m6A and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of m6A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m6A site in the HBV RNA and found that a conserved m6A consensus motif situated within the epsilon stem loop structure, is the site for m6A modification. The epsilon stem loop is located in the 3′ terminus of all HBV mRNAs and at both the 5′ and 3′ termini of the pgRNA. Mutational analysis of the identified m6A site in the 5′ epsilon stem loop of pgRNA revealed that m6A at this site is required for efficient reverse transcription of pgRNA, while m6A methylation of the 3′ epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m6A has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how m6A regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.

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Characterization of Nuclear RNases That Cleave Hepatitis B Virus RNA near the La Protein Binding Site

Journal of Virology ◽

10.1128/jvi.75.15.6874-6883.2001 ◽

2001 ◽

Vol 75 (15) ◽

pp. 6874-6883 ◽

Cited By ~ 45

Author(s):

Tilman Heise ◽

Luca G. Guidotti ◽

Francis V. Chisari

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Rna Binding ◽

Regulatory Element ◽

Murine Cytomegalovirus ◽

Mcmv Infection ◽

Stem Loop ◽

La Protein ◽

Nuclear Extracts ◽

B Virus

ABSTRACT Hepatitis B virus (HBV) RNA is downregulated by inflammatory cytokines induced in the liver by adoptively transferred HBV-specific cytotoxic T lymphocytes (CTLs) and during murine cytomegalovirus (MCMV) infections of the livers of HBV transgenic mice. The disappearance of HBV RNA is tightly associated with the cytokine-induced proteolytic cleavage of a previously defined HBV RNA-binding protein known as La autoantigen. La binds to a predicted stem-loop structure at the 5′ end of the posttranscriptional regulatory element of HBV RNA between nucleotides 1243 and 1333. In the present study, we searched for nuclear RNase activities that might be involved in HBV RNA decay. Nuclear extracts derived from control livers and CTL-injected and MCMV-infected livers were analyzed for the ability to cleave HBV RNA. Endonucleolytic activity that cleaved HBV RNA at positions 1269 to 1270 and 1271 to 1272, immediately 5′ of the stem-loop bound by the La protein (positions 1272 to 1293), was detected. Furthermore, we provide evidence that the cytokine-dependent downregulation of HBV RNA following MCMV infection is temporally associated with the upregulation of the endonucleolytic activity herein described. Collectively, these results suggest a model in which the steady-state HBV RNA content is controlled by the stabilizing influence of La and the destabilizing influence of nuclear RNase activities.

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Incorporation of Eukaryotic Translation Initiation Factor eIF4E into Viral Nucleocapsids via Interaction with Hepatitis B Virus Polymerase

Journal of Virology ◽

10.1128/jvi.01232-09 ◽

2009 ◽

Vol 84 (1) ◽

pp. 52-58 ◽

Cited By ~ 12

Author(s):

Seahee Kim ◽

Haifeng Wang ◽

Wang-Shick Ryu

Keyword(s):

Translation Initiation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Loop Structure ◽

Stem Loop ◽

Eukaryotic Translation ◽

Stem Loop Structure ◽

B Virus ◽

Rnp Complex ◽

Eukaryotic Translation Initiation

ABSTRACT The DNA genome of hepatitis B virus (HBV) replicates via reverse transcription within capsids following the encapsidation of an RNA template, the pregenomic RNA (pgRNA). We previously demonstrated that the 5′ cap proximity of the stem-loop structure (ε or epsilon), an encapsidation signal, is critically important for the encapsidation of the pgRNA (J. K. Jeong, G. S. Yoon, and W. S. Ryu, J. Virol. 74:5502-5508, 2000). Therefore, we speculated that the viral polymerase (Pol), while bound to the 5′ ε stem-loop structure, could recognize the cap via its interaction with eIF4E, a eukaryotic translation initiation factor. Our data showed the direct interaction between HBV Pol and eIF4E, as measured by coimmunoprecipitation. Further, we demonstrated that eIF4E interacts with the Pol-ε ribonucleoprotein complex (RNP) rather than Pol alone, resulting in eIF4E-Pol-ε RNP complex formation. In addition, we asked whether eIF4E remains engaged to the Pol-ε RNP complex during nucleocapsid assembly. Density gradient analysis revealed that eIF4E indeed was incorporated into nucleocapsids. It is of great importance to uncover whether the incorporated eIF4E contributes to viral reverse transcription or other steps in the HBV life cycle.

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Solution structure of the apical stem–loop of the human hepatitis B virus encapsidation signal

Nucleic Acids Research ◽

10.1093/nar/gkl582 ◽

2006 ◽

Vol 34 (16) ◽

pp. 4449-4457 ◽

Cited By ~ 32

Author(s):

Sara Flodell ◽

Michael Petersen ◽

Frederic Girard ◽

Janusz Zdunek ◽

Karin Kidd-Ljunggren ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Solution Structure ◽

Stem Loop ◽

Human Hepatitis ◽

Encapsidation Signal ◽

B Virus

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RNA-Binding Motif Protein 24 (RBM24) Is Involved in Pregenomic RNA Packaging by Mediating Interaction between Hepatitis B Virus Polymerase and the Epsilon Element

Journal of Virology ◽

10.1128/jvi.02161-18 ◽

2019 ◽

Vol 93 (6) ◽

Cited By ~ 3

Author(s):

Yongxuan Yao ◽

Bo Yang ◽

Yingshan Chen ◽

Hui Wang ◽

Xue Hu ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Rna Binding ◽

Binding Activity ◽

Hbv Infection ◽

Binding Motif ◽

Independent Manner ◽

Stem Loop ◽

Hbv Replication ◽

B Virus

ABSTRACTEncapsidation of pregenomic RNA (pgRNA) is a crucial step in hepatitis B virus (HBV) replication. Binding by viral polymerase (Pol) to the epsilon stem-loop (ε) on the 5′-terminal region (TR) of pgRNA is required for pgRNA packaging. However, the detailed mechanism is not well understood. RNA-binding motif protein 24 (RBM24) inhibits core translation by binding to the 5′-TR of pgRNA. Here, we demonstrate that RBM24 is also involved in pgRNA packaging. RBM24 directly binds to the lower bulge of ε via RNA recognition submotifs (RNPs). RBM24 also interacts with Pol in an RNA-independent manner. The alanine-rich domain (ARD) of RBM24 and the reverse transcriptase (RT) domain of Pol are essential for binding between RBM24 and Pol. In addition, overexpression of RBM24 increases Pol-ε interaction, whereas RBM24 knockdown decreases the interaction. RBM24 was able to rescue binding between ε and mutant Pol lacking ε-binding activity, further showing that RBM24 mediates the interaction between Pol and ε by forming a Pol-RBM24-ε complex. Finally, RBM24 significantly promotes the packaging efficiency of pgRNA. In conclusion, RBM24 mediates Pol-ε interaction and formation of a Pol-RBM24-ε complex, which inhibits translation of pgRNA and results in pgRNA packing into capsids/virions for reverse transcription and DNA synthesis.IMPORTANCEHepatitis B virus (HBV) is a ubiquitous human pathogen, and HBV infection is a major global health burden. Chronic HBV infection is associated with the development of liver diseases, including fulminant hepatitis, hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. A currently approved vaccine can prevent HBV infection, and medications are able to reduce viral loads and prevent liver disease progression. However, current treatments rarely achieve a cure for chronic infection. Thus, it is important to gain insight into the mechanisms of HBV replication. In this study, we found that the host factor RBM24 is involved in pregenomic RNA (pgRNA) packaging and regulates HBV replication. These findings highlight a potential target for antiviral therapeutics of HBV infection.

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